14738 • The Journal of Neuroscience, September 11, 2013 • 33(37):14738–14748

Development/Plasticity/Repair Ontogeny of Excitatory Spinal Neurons Processing Distinct Somatic Sensory Modalities

Yi Xu,1* Claudia Lopes,1* Hagen Wende,2 Zhen Guo,3 Leping Cheng,3 Carmen Birchmeier,2 and Qiufu Ma1 1Dana-Farber Cancer Institute and Department of Neurobiology, Harvard Medical School, Boston, Massachusetts 02115, 2Department of Neuroscience, Max Delbru¨ck Center, 13125 Berlin, Germany, and 3Institute of Neuroscience, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China

Spatial and temporal cues govern the genesis of a diverse array of neurons located in the dorsal spinal cord, including dI1-dI6, dILA , and dILB subtypes, but their physiological functions are poorly understood. Here we generated a new line of conditional knock-out (CKO) mice, in which the gene Tlx3 was removed in dI5 and dILB cells. In these CKO mice, development of a subset of excitatory neurons located in laminae I and II was impaired, including itch-related GRPR-expressing neurons, PKC␥-expressing neurons, and neurons expressing three neuropeptide genes: somatostatin, preprotachykinin 1, and the gastrin-releasing peptide. These CKO mice displayed marked deficits in generating nocifensive motor behaviors evoked by a range of pain-related or itch-related stimuli. The mutants also failed to exhibit escape response evoked by dynamic mechanical stimuli but retained the ability to sense innocuous cooling and/or warm. Thus, our studies provide new insight into the ontogeny of spinal neurons processing distinct sensory modalities.

Introduction from progenitors expressing the Olig3, The dorsal horn of the spinal cord processes diverse somatic sen- and (2) Lbx1-expressing class B neurons, derived from Olig3- sory information, including pain, itch, touch, cold, and warm negative progenitors (see Fig. 1A)(Gross et al., 2002; Mu¨ller et al., (Craig, 2003; Todd, 2010). Early electrophysiological and mor- 2002, 2005). Class B neurons are further divided into glutamater- phological studies suggest the existence of modality selective gic excitatory neurons (dI5 and dILB) marked by the expression spinal neurons (Christensen and Perl, 1970; Han et al., 1998), of the homeobox proteins Lmx1b and Tlx3, and GABAergic/ which is further supported by recent ablation and behav- glycinergic inhibitory neurons (dI4, dI6, and dILA) marked by the ioral studies (Sun et al., 2009; Mishra et al., 2012; Mishra and expression of the homeobox proteins Pax2 and Lhx2/9 (Gross et Hoon, 2013). For example, neurons expressing the gastrin- al., 2002; Mu¨ller et al., 2002; Qian et al., 2002; Helms and John- releasing peptide (GRPR) or the natriuretic polypep- son, 2003; Cheng et al., 2004, 2005; Glasgow et al., 2005; Rebelo et tide b receptor (Npra) are required to sense itch, but not pain al., 2010). (Sun et al., 2009; Mishra and Hoon, 2013). However, how Previously, we and others reported that the Tlx3 homeobox modality-selective spinal neurons emerge during develop- gene is necessary for proper development of dI3, dI5, and dILB ment is still poorly understood. excitatory neurons in the dorsal spinal cord, including specifica- Early during development, eight groups of molecularly dis- tion of the glutamatergic and peptidergic transmitter tinct dorsal horn neurons have been identified, namely, dI1-dI6, (Qian et al., 2002; Cheng et al., 2004; Xu et al., 2008; Guo et al., dILA, and dILB (Gross et al., 2002; Mu¨ller et al., 2002; Helms and 2012). A recent study shows that class A dI3 neurons are involved Johnson, 2003) (see Fig. 1A). Based on differential expression of with sensory motor coordination, such as hand grasp (Bui et al., the homeodomain protein Lbx1, they are divided into (1) Lbx1- 2013). The focus of this study, however, is the physiological func- negative class A excitatory neurons (dI1–3), which are derived tions of class B dI5 and dILB neurons, which are still unknown. Genetic fate mapping shows that the majority of Tlx3 lineage neurons are enriched in superficial laminae (I-III), although also Received Nov. 29, 2012; revised Aug. 3, 2013; accepted Aug. 7, 2013. scattered throughout the ventral laminae (Xu et al., 2008). Be- Author contributions: Y.X., C.L., Z.G., L.C., and Q.M. designed research; Y.X., C.L., Z.G., L.C., and Q.M. performed research; H.W. and C.B. contributed unpublished reagents/analytic tools; Y.X., C.L., Z.G., L.C., and Q.M. analyzed cause dI3 neurons are enriched in the deep dorsal horn (Helms data; Y.X., C.L., L.C., C.B., and Q.M. wrote the paper. and Johnson, 2003; Bui et al., 2013), it can be certain that those The work done in the Q.M. laboratory was supported by the National Institutes of Health, National Institute of Tlx3 lineage neurons located in the superficial laminae must be Neurological Disorders and Stroke Grant R01NS047710. We thank Dr. Silvia Arber for the Tau-loxp-STOP-lox-mGFP- derived from dI5 and dIL neurons. Superficial laminae normally IRES-NLS-LacZ reporter mice; Dr. Jean-Francois Brunet for the Phox2a antibody; and Drs. Clifford Woolf, Wendy B Knowlton, Fu-Chia Yang, and Bo Duan for critical discussions and comments. receive inputs from primary sensory afferents that transmit The authors declare no competing financial interests. pain-, itch-, and temperature-related sensory information *Y.X. and C.L. contributed equally to this work. (Todd, 2010). To determine to what degree dI5 and dILB neurons Correspondence should be addressed to Dr. Qiufu Ma, Dana-Farber Cancer Institute and Department of Neuro- process these types of somatic sensory information, here we used biology, Harvard Medical School, 1 Jimmy Fund Way, Boston, MA 02115. E-mail: [email protected]. DOI:10.1523/JNEUROSCI.5512-12.2013 Lbx1-Cre mice (Sieber et al., 2007) to selectively remove Tlx3 in Copyright © 2013 the authors 0270-6474/13/3314738-11$15.00/0 these neurons. Subsequent histochemical and behavioral analy- Xu, Lopes et al. • Excitatory Spinal Neurons and Somatic Sensory Modalities J. Neurosci., September 11, 2013 • 33(37):14738–14748 • 14739 ses provide new insight into the ontogeny of spinal neurons pro- Capsaicin was intradermally administrated in a dosage of 2.5 ␮g/10 ␮l cessing distinct types of somatic sensory modalities. into the right hindpaw, and the duration of lifting, licking, and flinching was measured. To measure sensitivity to noxious cold, the cold plate Materials and Methods (IITC) was set to 0°C. The mice were video-recorded for 120 s, and the Animals. The generation of mice carrying the Tlx3 conditional null allele combined number of hindpaw flicking, licking, or jumping was (Tlx3F/ϩ), the Lbx1cre mouse line, the Tlx3Cre mice, and the Tau-loxp- measured. STOP-lox-mGFP-IRES-NLS-LacZ reporter line had been described pre- For the temperature chamber assay, animals were allowed to explore viously (Hippenmeyer et al., 2005; Sieber et al., 2007; Xu et al., 2008; adjacent surfaces, with one held at 20°C and the other ranging from 30°C Lopes et al., 2012). In all timed matings, the morning that vaginal plugs to 5°C. The mice were video-recorded for 5 min, and the percentages of were observed was considered to be E0.5. Genotypes were identified by time staying at 20°C chamber were determined. The acetone evaporation PCR analysis of genomic DNA extracted from mouse tail tissue, and the cooling assay was performed as previously described (Knowlton et al., following PCR primers were used to identify the floxed and deleted Tlx3 2011) with some modifications. Mice were acclimated for 10 min in an alleles:5Ј-TGTTTCGCCTCCTTTGCTCG-3Јand5Ј-GTTGGATGGAAG elevated wire grid. A syringe with a piece of rubber tubing attached to the CAAAGATAG-3Ј. For histochemical studies, mice at P28 or younger end was filled with acetone and the plunger depressed so that a small drop ages were used. For behavioral analyses, mutant and control littermates of acetone formed at the top of the tubing. The syringe was raised to the at P21-P28 were used. All animal procedures are contained in protocols hindpaw from below, depositing the acetone drop on the paw. The test reviewed and approved by the Animal Care and Use Committees at the was repeated for 10 times (5 times for each paw), with intervals of 3 min. Dana-Faber Cancer Institute. Both males and females were used for the Responses were video-recorded, and duration times of lifting, licking, studies. flinching, shacking, and rotating on the torso were determined. To mea- In situ hybridization and immunostaining. Detailed methods for single- sure punctate mechanical sensitivity, we placed animals on an elevated color in situ hybridization and in situ hybridization combined with flu- wire grid and the lateral plantar surface of the hindpaw stimulated with orescent immunostaining had been described previously (Liu et al., calibrated von Frey monofilaments (0.0174–4.57 g). The paw with- 2010). The following in situ probes were described previously, including drawal threshold for the von Frey assay was determined using Dixons SOM, Tac1, VGLUT2, and GRPR (Cheng et al., 2004; Xu et al., 2008). up-down method (Chaplan et al., 1994). To measure dynamic mechan- GRP in situ probe was amplified with gene-specific sets of PCR prim- ical sensitivity, the middle part of the right hindpaw was stimulated by ers and cDNA template prepared from postnatal day 0 (P0) mouse light stroking with a cotton swab, in the direction from heel to toe. The spinal cords with a final fragment size of 0.387 kb. The following test was repeated three times, with intervals of 10 seconds. For each test, antibodies were used for single or double immunostaining: rabbit a score of 0 indicates no movement, and a score of 1 is a lifting of the anti-Pax2 (1:500, Zymed Laboratories), rabbit anti-VGLUT1 (1:1000, stimulated paw and/or walking way. For each mouse, the accumulative Swant), rabbit anti-phospho-ERK (1:250, Cell Signaling Technol- scores of three tests were used to indicate “the dynamic score” shown in ogy), rabbit anti-Pkc␥ (1:500, Santa Cruz Biotechnology), and chick Figure 7A. anti-lacZ (1:500, Abcam). The rabbit anti-Tlx3 (1:500) and guinea pig To measure sensorimotor coordination, we performed the rotarod test. anti-Tlx3 (1:500) were acquired from C. Birchmeier at Max- Initially, mice were habituated for 1 min at a rotating speed of 4.0 rpm/s. For Delbruck-Center for Molecular Medicine, Berlin, Germany, and rab- the actual test, we set the ramp to start at a velocity of 4.0 rpm/s, with a bit anti-Phox2a was a gift from Dr. Jean-Francois Brunet at Centre continuous acceleration of 0.4 rpm/s. The time points when mutant and National de la Recherche Scientifique, Paris, France. Sections were control mice fell off the ramp were recorded. Data for the different behav- Ϯ processed with immunofluorescence by incubating overnight with ioral assays are represented as the mean SE. Statistical significance was Ͻ primary antibody and1hatroom temperature with appropriate assessed with the Student’s t test, with p 0.05 considered significant. The fluorescence-conjugated secondary antibodies (Invitrogen). experimenter was blinded to the genotype of animals. Counting p-ERKϩ cells. To count p-ERK ϩ spinal cells induced by heat, four pairs of P28 control and mutant mice were used, and the right Results hindpaw of each of these mice was dipped into a 50°C water bath for 20 s F/F Cre/؉ while holding the mouse by its neck, tail, and the left paw. Five minutes Generation of Tlx3 ;Lbx1 conditional knock-out mice later, the mouse was killed and perfused transcardially with 4% parafor- Tlx3 null mice die at birth (Shirasawa et al., 2000). We recently maldehyde in 0.1 M phosphate buffer, pH 7.4. The lumbar spinal cord was generated a mouse line carrying a conditional null allele of Tlx3 F/ϩ removed from L3 to L6, and transverse sections (20 ␮m thickness) were (Lopes et al., 2012), referred to as Tlx3 . To study the physio- cut and processed for p-ERK immunohistochemistry. Cells with a clear logical functions of class B dI5 and dILB neurons, we crossed nuclear and staining levels clearly above background were Tlx3F/ϩ mice with Lbx1-Cre mice (Sieber et al., 2007) to generate ϩ counted. The total number of p-ERK cells per set of sections through Tlx3F/F;Lbx1Cre/ϩ mice, referred to here as conditional knock-out the L3-L6 spinal cord was determined, and values were presented as (CKO) mice. As indicated by developmental ontogeny of spinal mean Ϯ SD. The differences between control and mutant samples were Ͻ neurons (Fig. 1A), in these CKO mice, Tlx3 was removed selec- subjected to a Student’s t test, with p 0.05 considered significant. tively in Lbx1-expressing class B dI5 and dIL neurons (Fig. 1A). Behavioral analysis. All pain and itch behavioral tests were performed B as previously described (Liu et al., 2010). All animals (Tlx3F//F;Lbx1cre In wild-type mice, expression of both Tlx3 and Lbx1 is initiated mutants and Tlx3F/F or Tlx3F/ϩ control littermates) were acclimatized to immediately in newly formed postmitotic neurons (Gross et al., the behavioral testing apparatus for at least three ‘‘habituation” sessions. 2002; Mu¨ller et al., 2002; Qian et al., 2002). We found that Tlx3 Two days before the injection, the nape of the neck was shaved after brief expression in the dorsal spinal cord was still detected at E12.5 in anesthesia with isoflurane (2% in 100% oxygen). A total of 10 ␮gof CKO mice (Fig. 1B) but was largely eliminated by E16.5 (data not Compound 48/80 (Sigma-Aldrich), 100 ␮g of PAR2 agonist SLIGRL- shown). At P21, persistent Tlx3 expression, which was detected in NH2 (Bachem), or 200 ␮g of chloroquine (Sigma-Aldrich) in 50 ␮lof superficial dorsal horn laminae in wild-type mice (Fig. 1C, ar- sterile saline were injected intradermally into the nape, and a camcorder row), was not detected in CKO mice (Fig. 1C), suggesting that (Sony model DCR-SR220) was positioned to video-record the behavior Tlx3-persistent neurons belong to Lbx1 lineage neurons. This is of mice. The video recording was played back and scratching bouts were consistent with class B dorsal horn excitatory neurons (dI5/dIL ) counted. The experimenter was blinded to the genotype of the animals, B and only the bouts to the shaved region were counted. To measure heat being defined by the expression of the homeobox protein Lmx1b (Gross et al., 2002; Mu¨ller et al., 2002), and persistent Tlx3 being sensitivity, we placed mice on a hot plate (IITC) and the latency to hind- ϩ paw flicking, licking, or jumping was measured. The hot plate was set to confined to these Lmx1b spinal neurons (Rebelo et al., 2010). 50°C with a cutoff time of 60 s, or to 54°C with a cutoff time of 30 s. All We found that CKO mice survived to postnatal stages (see animals were tested sequentially with a minimum of 5 min between tests. below). In the hindbrain, Tlx3 is expressed transiently in, but 14740 • J. Neurosci., September 11, 2013 • 33(37):14738–14748 Xu, Lopes et al. • Excitatory Spinal Neurons and Somatic Sensory Modalities necessary for proper development of, sev- eral Lbx1-negative class A neurons that are involved in respiration control, in- cluding noradrenergic (NA) neurons and the nucleus of the solitary tract (NTS) in the hindbrain (Qian et al., 2001, 2002; Sieber et al., 2007). Development of these Lbx1-negative neurons was naturally un- affected in CKO mice because Lbx1-Cre was used for making conditional knock- outs, as indicated by the normal expres- sion of (1) the homeobox gene Phox2b,a marker for NTS neurons, and (2) the do- pamine ␤ hydroxylase, a marker for NA neurons (data not shown) (Qian et al., 2001). In Tlx3 complete null mice, the loss of NA and NTS neurons causes a failure in central respiration control and neonatal lethality (Shirasawa et al., 2000; Qian et al., 2001). Accordingly, the preservation of these neurons in CKO mice explains why these mutants survive.

Selective impairment of lamina I/II neurons in CKO mice Tlx3 is expressed persistently in neurons lo- cated in the superficial dorsal horn, but transiently in more ventral laminae (Xu et al., 2008). With the preservation of early Tlx3 expression in CKO mice, we hypothe- sized that the development of Tlx3- persistent neurons might be preferentially impaired. Before we tested this hypothesis, we first examined which laminae contain Tlx3-persistent neurons. VGLUT1 is the vesicular glutamate transporter expressed in peripheral low threshold myelinated mechanoreceptors and proprioceptors that terminate in the inner layer of lamina II and more ventral laminae (Li et al., 2003). Dou- ble immunostaining showed that the ven- tral portion of Tlx3-persistent neurons was intermingled with most dorsally localized ϩ Figure 1. Ontogeny of spinal dorsal horn neurons and generation of CKO mice. A, Schematics showing the ontogeny of eight VGLUT1 terminals (Fig. 2A), and as de- ϩ ϩ(T/P) ϩ distinct groups of dorsal horn neurons (dI1–6, dILA, dILB). Tlx3 (T), Neurons expressing Tlx3 transiently; Tlx3 , neurons scribed below, corresponded to PKC␥ expressing Tlx3 transiently or persistently. B, C, Tlx3 immunostaining on transverse spinal sections at indicated stages and geno- neurons that represent the inner layer of types. At E12.5, Tlx3 protein expression was comparable between control (Ctrl) and CKO embryos (B, arrows). At P21, persistent lamina II (Neumann et al., 2008). Thus, Tlx3 expression was observed in the superficial dorsal horn of Ctrl mice (C, arrow) but abolished in CKO mice (C). Tlx3-persistent neurons are confined to laminae I and II. (Fig. 2D, arrowheads) (Xu et al., 2008). Here, we found a late We then found that development of lamina I/II neurons was wave of Tac1 ϩ neurons that emerged during postnatal develop- compromised in CKO mice. The somatostatin neuropeptide ment and were located in lamina II (Fig. 2D, arrow). In CKO gene (SOM) is expressed in both excitatory and inhibitory neu- mice, whereas the late wave of Tac1 expression in lamina II was rons, with Tlx3-dependent SOM ϩ excitatory neurons located in eliminated (Fig. 2D, arrows), the early wave in deep laminae was the superficial laminae (Xu et al., 2008). Double staining of SOM at least partially preserved (Fig. 2D, arrowheads). Thus, the mRNA and VGLUT1 showed that SOM expression was elimi- ϩ ϩ nated in the P21 CKO superficial dorsal horn but still detected in SOM and Tac1 subsets of excitatory neurons that are located more ventral laminae (Fig. 2B). Loss of SOM expression occurred at laminae I and II are selectively compromised in CKO mice. Because Tac1 expression is eliminated in Tlx3 complete null by E16.5 (data not shown) and at both lumbar (Fig. 2B) and ϩ cervical (Fig. 2C) levels at P21. The preprotachykinin gene (Tac1) mice (Xu et al., 2008), the preservation of a subset of Tac1 encodes two neuropeptides: substance P and neurokinin A (Ho¨k- neurons in CKO mice suggests that the development of some felt, 1991). There are two waves of Tac1 expression in the dorsal Tlx3-transient deep dorsal horn is unaffected in CKO mice. To spinal cord. We reported previously that the early wave is estab- further support this, we examined additional markers. A subset of lished at embryonic stages and enriched in the deep dorsal horn Tlx3-transient dI5 neurons is marked by the expression of Xu, Lopes et al. • Excitatory Spinal Neurons and Somatic Sensory Modalities J. Neurosci., September 11, 2013 • 33(37):14738–14748 • 14741

Figure 2. Selective loss of markers in laminae I/II of CKO mice. A, Double immunostaining of Tlx3 protein in spinal neurons (green) with VGLUT1 protein in primary afferents (red) on a transverse lumbar spinal section from a P21 wild-type mouse. B, Double staining of the VGLUT1 protein in primary afferent terminals (green) and cytoplasmic SOM mRNA in spinal neurons (pseudo-red, by in situ hybridization) on transverse P21 lumbar spinal cord sections of control (Ctrl) and CKO mice. There is a selective loss of SOM mRNA in lamina I/II dorsal to VGLUT1 ϩ terminals (arrow). C, SOM in situ hybridization on transverse P21 spinal sections at the cervical level. Arrow indicates SOM expression in the superficial laminae. D, Tac1 in situ hybridization on transverse P21 lumbar spinal cord sections. Arrows and arrowheads indicate Tac1 expression in superficial and deep laminae, respectively. E, Phox2a in situ hybridization on E16.5 lumbar spinal cord sections. F–H, Double staining ofthenLacZprotein(green)andSOMmRNA(F,red),Tac1mRNA(G,red),orPhox2aprotein(H,red)onP21orP0lumbarspinalcordsectionsfromTau-LSL-nlacZ;Lbx1-Crefatemappingmice.Arrows and arrowheads indicate double or singular staining, respectively. I, VGLUT2 in situ hybridization on E16.5 lumbar spinal sections with indicated genotypes.

Phox2a (Qian et al., 2002). We again found that these Phox2a ϩ neurons originate mainly from Lbx1 ϩ class B neurons, but neurons showed normal ventral migration in CKO mice at E16.5 also to a lesser degree from Lbx1 Ϫ class A neurons. In addi- (Fig. 2E) and at P0 (data not shown). Development of Lbx1- tion, most, if not all, Tac1 ϩ neurons, as well as all Phox2a ϩ negative class A spinal neurons is naturally unaffected. For neurons coexpressed nlacZ (Fig. 2G,H), suggesting that these example, dI1 neurons, marked by the expression of BarhL1 neurons are also derived from Lbx1 lineage class B neurons. (Bermingham et al., 2001; Ding et al., 2012), and dI3 neurons, This finding suggests that the normal development of these marked by the expression of Islet1 (Gross et al., 2002; Mu¨ller et Tlx3-transient deep dorsal horn neurons in CKO mice is the al., 2002; Qian et al., 2002), all showed normal ventral migra- result of the preservation of transient Tlx3 expression in class tion at E16.5 and P0 in CKO mice compared with controls B neurons, rather than a separate developmental origin from (data not shown). Lbx1 Ϫ class A neurons. The incomplete loss of SOM ϩ and Tac1 ϩ neurons, and Tlx3 is known to determine glutamatergic over GABAergic the normal development of Phox2a ϩ neurons, does raise a neurotransmitter phenotypes, and VGLUT2 expression in the question as to whether or not these neurons originate fully lumbar superficial dorsal horn is eliminated in Tlx3 null mice from Lbx1 ϩ class B neurons. To address this, we crossed Lbx1- (Cheng et al., 2004). Interestingly, with the preservation of early Cre mice (Sieber et al., 2007)withtheTau-loxp-STOP-lox- Tlx3 expression in CKO mice, VGLUT2 expression was largely mGFP-IRES-NLS-LacZ reporter mice (Hippenmeyer et al., unaffected at E16.5 (Fig. 2I). As a comparison, SOM expression 2005) to generate mice referred to here as Tau-LSL-nlacZ; in superficial laminae was already eliminated by E16.5 (data not Lbx1-Cre. In these mice, the Lbx1 lineage neurons are marked shown). Thus, transient Tlx3 expression is sufficient to establish by the expression of the nuclear lacZ protein, regardless of the glutamatergic transmitter , which is in contrast persistent or transient Lbx1 expression. Double staining with the requirement of Tlx3 activity beyond E12.5 in establish- showed that most, but not all, SOM ϩ neurons coexpressed ing other molecular identities, such as the expression of SOM and nLacZ (Fig. 2F, arrow vs arrowhead), suggesting that SOM ϩ Tac1 in superficial laminae. 14742 • J. Neurosci., September 11, 2013 • 33(37):14738–14748 Xu, Lopes et al. • Excitatory Spinal Neurons and Somatic Sensory Modalities

dorsal horn results in reduced innervation by CGRP ϩ and IB4 ϩ afferents, but the basic organization of these terminals in the dorsal horn is preserved in CKO mice.

Impaired behavioral responses evoked by pain-related stimuli in CKO mice CKO mice can only survive for 1–2 months. These mutant mice showed overgrowth of teeth, possibly leading to malocclusion that may affect feeding (Fig. 4A), but the underlying cause is not known. Nonetheless, CKO mice at 3–4 weeks of age grossly looked healthy, although their body sizes were smaller than wild- type littermates (data not shown). We therefore performed all behavioral analyses at these ages, with Tlx3F/F or Tlx3F/ϩ litter- mates as controls. CKO mice showed normal proprioception and sensorimotor coordination, as suggested by proper clustering of the forelimbs and the extension of hindlimbs when suspended by the tail (Fig. 4B). Furthermore, after one round of training, CKO and control littermates remained on an accelerating rotarod for similar amounts of time (Fig. 4C). We next applied a mechanical stimulus to the plantar surface of the hindpaw using von Frey fibers and determined the thresh- old leading to hindpaw lifting and flinching. In comparison with controls, CKO mice showed a marked increase in withdrawal thresholds, suggesting a deficit in generating proper reflex behav- ior in response to noxious mechanical stimuli (Fig. 4D). To measure behavioral response to noxious heat, we placed mice on a hot plate set to 50°C or 54°C and found that the latency Figure 3. Normal DRG neuron development and reduced central innervation in CKO mice. of hindpaw lifting/flinching increased markedly in CKO mice, A–E, In situ hybridization with indicated probes on transverse sections through lumbar DRG of suggesting a defect in processing heat-related sensory informa- P21 control (Ctrl) and CKO mice. F, Double staining on transverse P21 lumbar spinal cord sec- tion (Fig. 4E,F). Heat pain is mediated by DRG neurons express- tions with CGRP immunostaining (red) and IB4 binding (green). ing the transient receptor potential channel TRPV1 (Cavanaugh et al., 2009; Mishra and Hoon, 2010), which is also the receptor Reduced innervation by primary sensory afferents in for the chili pepper ingredient capsaicin (Caterina et al., 1997). CKO mice Consistently, intraplantar capsaicin injection, which evoked ro- Dorsal horn laminae I and II are mainly innervated by sensory bust licking and flinching in controls, failed to elicit responses in neurons detecting pain-, itch-, and temperature-related sensory CKO mice (Fig. 4G). information (Lallemend and Ernfors, 2012). These sensory neu- To more directly assess how spinal neurons responded to rons, located in the DRG, are divided into two main subtypes, painful stimuli in CKO mice, we took advantage of the previous peptidergic and nonpeptidergic neurons. Peptidergic neurons finding that noxious painful stimuli, such as 50°C heat, activate express the calcitonin gene-related peptide (CGRP) and the neu- the ERK protein kinase through phosphorylation (p-ERK) selec- rotrophin receptor TrkA, and innervate lamina I and the outer tively in lamina I/II neurons (Ji et al., 1999). We found that, after layer of lamina II. Many nonpeptidergic neurons can be labeled 50°C heat stimulation of the CKO hindpaw for a short period (20 by the binding of isolectin B4 (IB4), coexpress the neurotrophin s), the number of p-ERK ϩ neurons in superficial laminae of the receptor Ret, and innervate the dorsal portion of the inner layer lumbar spinal cord was greatly reduced compared with controls, of lamina II. Deep dorsal horn laminae (III-V) are innervated from 190 Ϯ 44 per set of sections in control mice to 31 Ϯ 9in mainly by myelinated mechanoreceptors marked by the expres- CKO mice (n ϭ 4, p Ͻ 0.05), an 84% reduction (Fig. 4H,I). sion of various neurotrophin receptors, including TrkC, TrkB, Together, these data suggest that Tlx3-dependent dI5 and/or dILb and Ret (Lallemend and Ernfors, 2012), as well as by the expres- neurons are required to process mechanical and heat pain-related sion of VGLUT1 (Li et al., 2003). In CKO mice, DRG neuron sensory information. development and survival were unaffected, as suggested by the grossly normal expression of TrkA, TrkB, TrkC, CGRP, and Ret Reduced scratching response evoked by pruritic compounds (Fig. 3A–E). This is consistent with the lack of Lbx1 expression in in CKO mice DRG (Gross et al., 2002; Mu¨ller et al., 2002), and unaffected Tlx3 To measure itch-related behavior, we performed nape injections expression in Tlx3F/F;Lbx1Cre/ϩ CKO mice (data not shown). of itch-inducing compounds and monitored scratching re- Despite normal differentiation of DRG neurons, we found sponse. Compound 48/80 activates a histamine-dependent itch that there are much fewer CGRP ϩ and IB4 ϩ nerve terminals in pathway (Sugimoto et al., 1998), and the number of scratch bouts CKO dorsal spinal cord, although their relative dorsoventral pro- evoked by this compound was reduced, but not completely elim- jection pattern remained (Fig. 3F). In contrast, innervation of inated, in CKO mice compared with controls (Fig. 5A). Both the VGLUT1 ϩ low threshold mechanoreceptors to the deep dorsal malaria drug chloroquine and the Par2 agonist peptide SLIGRL- horn and to the ventral motor neurons was largely unaffected NH2 evoke histamine-independent itch by activating the (data not shown). Thus, developmental impairment of a large G-protein coupled receptors Mrgpra3 and Mrgprc11, respec- subset of Tlx3-dependent excitatory neurons in the superficial tively (Liu et al., 2009, 2011; Wilson et al., 2011). Nape injection Xu, Lopes et al. • Excitatory Spinal Neurons and Somatic Sensory Modalities J. Neurosci., September 11, 2013 • 33(37):14738–14748 • 14743

Figure4. Longteeth,normalsensorimotorcoordinationandimpairednocifensivebehaviorsinCKOmice.A,MalocclusioninaP28CKOmouse(arrows)comparedwithacontrol(Ctrl).B,P28CKO mice exhibiting normal sensorimotor coordination. C, Rotarod assay. No significant difference was found between Ctrl and CKO mice on time (seconds) staying on accelerating rotarod. Ctrl, n ϭ 10, 45.6 Ϯ 5.5 s; CKO, n ϭ 10, 39.4 Ϯ 5.2 s. p ϭ 0.44. D, von Frey test. CKO mice showed a higher withdrawal threshold than Ctrl mice. Ctrl, n ϭ 7, 0.29 Ϯ 0.04 g; CKO, n ϭ 7, 1.36 Ϯ 0.05 g. ***p Ͻ 0.001. E, F, Hot plate assay. CKO mice showed longer latency (seconds) in response to both 50°C and 54°C stimuli compared with Ctrl mice. E, For 50°C: Ctrl, n ϭ 8, 23.4 Ϯ 1.2 s; CKO, n ϭ 8, 56.5 Ϯ 1.0 s. ***p Ͻ 0.001. F, For 54°C: Ctrl, n ϭ 6, 7.6 Ϯ 1.0 s; CKO, n ϭ 6, 26.8 Ϯ 1.3 s. **p Ͻ 0.01. G, Capsaicin hindpaw injection assay: measuring the duration (seconds) of licking and flinching. Abolished response in CKO mice. Ctrl, n ϭ 9, 31.8 Ϯ 3.2 s; CKO, n ϭ 9, 0.0 Ϯ 0.0 s. H, p-ERK immunostaining on P28 lumbar spinal transverse sections after heat stimulation. Arrows indicate superficial laminae. I, Quantitative analysis of p-ERK ϩ neurons per set of sections through L3–L6. Ctrl, n ϭ 4, 190 Ϯ 44; CKO, n ϭ 4, 31 Ϯ 9. *p Ͻ 0.05. Error bars indicate SEM.

of these compounds evoked robust scratching responses in con- Impairment of touch-evoked escape responses in CKO mice trol littermates, but little in CKO mice. Thus, Tlx3-dependent dI5 In response to the dynamic mechanical stimulus of stroking the and/or dILb class B excitatory neurons are required to process hindpaw with a cotton swab, control mice responded by lifting itch-related information (Fig. 5A). the hindpaw and walking away. Interestingly, this touch-evoked Dorsal horn neurons expressing GRPR, the receptor for the “escape” response was virtually abolished in CKO mice (Fig. 6A). GRP, have been implicated in itch sensing (Sun et al., 2009). We To assess the potential cause for this behavioral deficit, we next found that expression of both GRPR and GRP was eliminated in examined neurons expressing the ␥ isoform of PKC␥ in the inner CKO mice (Fig. 5B). lamina II, which respond to dynamic mechanical stimuli (Mirau- Double staining on spinal sections of Tau-LSL-nlacZ;Lbx1- court et al., 2007; Neumann et al., 2008) and belong to excitatory Cre fate-mapping mice showed that neurons with detectable neurons (Polga´r et al., 1999). We found that PKC␥ expression in GRPR or GRP mRNA coexpressed nLacZ (Fig. 5C,D), suggesting the inner lamina II was eliminated in CKO mice (Fig. 6B). As a that GRPR ϩ and GRP ϩ neurons represent subsets of Lbx1 ϩ class positive control, PKC␥ ϩ fibers in the dorsal funiculus were still B neurons. To further investigate the ontogeny of GRPR ϩ neu- detected in both control and mutant mice (Fig. 6B). rons, we crossed Tlx3-Cre mice with the Tau-LSL-nLacZ reporter Consistently, double staining on spinal sections of Tau-LSL- mice, with Tlx3 lineage spinal neurons permanently marked by nlacZ;Lbx1-Cre fate-mapping mice showed that all PKC␥ ϩ neu- the expression of nlacZ (Hippenmeyer et al., 2005; Xu et al., rons coexpressed nlacZ (Fig. 6C), suggesting that they represent 2008). Double staining showed that GRPR ϩ neurons are con- Lbx1 lineage class B neurons. Furthermore, many, although not fined to Tlx3 lineage excitatory neurons (Fig. 5E). Consistently, all, PKC␥ ϩ neurons showed persistent Tlx3 expression (Fig. 6D). GRPR ϩ neurons did not express Pax2 (Fig. 5F), which is a The location of PKC␥ ϩ neurons delineates the ventral border of marker for inhibitory neurons (Cheng et al., 2004). Collectively, Tlx3-persistent neurons (Fig. 6D). We hypothesized that the im- these studies suggest that Tlx3 may autonomously control the paired development of PKC␥ ϩ mechanoresponsive cells might development of itch-related GRPR ϩ neurons. underlie the loss of the escape response evoked by dynamic me- 14744 • J. Neurosci., September 11, 2013 • 33(37):14738–14748 Xu, Lopes et al. • Excitatory Spinal Neurons and Somatic Sensory Modalities

Figure5. ImpairedscratchingresponseevokedbypruriticcompoundsinCKOmice.A,Scratchingresponsewasexaminedinanimalsat3to4weeksold.Napeinjectionsofcompound48/80(10␮g)evoked reducedscratchingboutsinCKOmicecomparedwithcontrols(Ctrl).Ctrl,nϭ6,149Ϯ74;CKO,nϭ6,21Ϯ19.**pϽ0.01.InjectionofthePAR2agonistSLIGRL-NH2(100␮g)evokedvirtuallynoscratching inCKOmice.Ctrl,nϭ6,134Ϯ111;CKO,nϭ6,1Ϯ2.*pϽ0.05.Injectionofchloroquineinjection(200␮g)alsodidnotinducescratchinginmutants.Ctrl,nϭ4,152Ϯ70;CKO,nϭ4,0Ϯ0.*pϽ0.05. Error bars indicate SEM. B,In situ hybridization on P28 lumbar spinal cord sections with indicated probes. C,D, GRPR ϩ and GRP ϩ neurons are derived from Lbx1 lineage neurons. Double staining of the nLacZ protein (green) and indicated mRNA (pseudo-red) on P21 lumbar spinal cord sections from Tau-LSL-nlacZ;Lbx1-Cre fate mapping mice. Arrows indicate colocalization. E, GRPR ϩ neurons are derived from Tlx3 lineage neurons. Double staining of the nLacZ protein (green) and GRPR mRNA (pseudo-red) on P0 lumbar spinal cord sections of Tau-LSL-nlacZ;Tlx3-Cre fate mapping mice. Arrows indicate colocalization. F, Double staining of the nuclear Pax2 protein (green) and GRPR mRNA (pseudo-red) on an E16.5 wild-type spinal section, showing no colocalization (arrowhead). chanical stimuli in CKO mice, although future studies are needed 15°C or 5°C chamber (Fig. 7C). The normal selection of 30°C to test this hypothesis. over 20°C chambers suggests that CKO mice are able to sense innocuous cooling and/or warm. In the 20°C versus 5°C assay, CKO mice are able to sense innocuous cooling and/or warm, some investigators used the avoidance of the 5°C chamber as a but not noxious cold way of measuring noxious cold sensation (Bautista et al., 2007; We next examined how mutants responded to cold stimuli. We Colburn et al., 2007; Dhaka et al., 2007; Knowlton et al., 2010). first placed controls and mutants onto the cold plate set to 0°C for However, both mutant and control mice spent ϳ94% of time in 2 min. We found that most control mice exhibited paw lifting and the 20°C chamber (Fig. 7C): the moment they reached the 5°C licking, but these nocifensive responses evoked by noxious cold chamber, they immediately withdrew to the warmer side (data were virtually abolished in CKO mice (Fig. 7A). We next per- not shown). Such momentary exposure to the cold chamber un- formed the acetone evaporation assay, which cools skin temper- likely drops the skin temperature to the noxious cold range. Thus, ature down to 14–18°C, around the transition zone of noxious the normal selection of 20°C over 5°C by CKO mice most likely versus innocuous cold temperatures (Colburn et al., 2007). We reflects a normal sense of innocuous cooling, rather than cold found that both CKO mice and control littermates showed sim- pain, particularly considering that these mice fail to generate ilar responses to the evaporative cooling as measured by the du- pain-suggestive responses at 0°C (Fig. 7A). Collectively, these ration of paw shaking, lifting, and licking (Fig. 7B), suggesting findings suggest that CKO mice fail to respond to extreme cold that Tlx3 CKO mice appear to retain the ability to sense innocu- but retain the ability to sense innocuous cooling and/or warm. ous or mild noxious cold. To further explore whether CKO mice were able to sense in- Discussion nocuous cold, we performed the temperature chamber selection Temporal control of dorsal horn excitatory assay (Bautista et al., 2007; Colburn et al., 2007; Dhaka et al., neuron phenotypes 2007; Knowlton et al., 2010). We found that mutant and control Tlx3 and its related gene Tlx1 act as selector genes that coordinate mice were indistinguishable in choosing the 30°C over the room the development of a diverse array of dorsal horn excitatory neu- temperature (ϳ20°C) chamber, or the 20°C chamber over the rons by specifying glutamatergic and peptidergic transmitter Xu, Lopes et al. • Excitatory Spinal Neurons and Somatic Sensory Modalities J. Neurosci., September 11, 2013 • 33(37):14738–14748 • 14745

Figure 7. Cold behavior analyses. A, The 0°C cold plate assay. The numbers of licking/flinch- ing (“L/F”) were counted. The CKO mice failed to respond to noxious cold (Ctrl, n ϭ 20, 7.45 Ϯ 0.13;CKO,nϭ5,1.27Ϯ0.08).***pϽ0.001.B,Theacetoneevaporationassay.Nodifference between control and CKO mice (Ctrl, n ϭ 7, 8.73 Ϯ 0.97; CKO, n ϭ 5, 6.40 Ϯ 0.57). p Ͼ 0.05. C,Thetemperaturechamberselectionassay.Thepercentagesoftimestayingatthe20°Ccham- ber were determined. No differences between control and CKO mice were observed at four different sets of temperatures. For 20°C versus 20°C selection chamber: control, n ϭ 6, 52.8 Ϯ Figure 6. Impairment of touch-evoked escape responses and Pkc␥ ϩ neurons in CKO mice. 6.8; CKO, n ϭ 6, 52.9 Ϯ 56.0. p ϭ 0.8. For 20°C versus 30°C: control, n ϭ 6, 18.6 Ϯ 6.4; CKO, A, The cotton wipe assay. The dynamic score, used to measure touch-evoked escapes, was n ϭ 6, 18.4 Ϯ 14.5. p ϭ 0.9. For 20°C versus 15°C: control, n ϭ 6, 76.9 Ϯ 11.0; CKO, n ϭ 6, reduced in CKO mice compared with control littermates (Ctrl). Ctrl, n ϭ 7, 2.71 Ϯ 0.18; CKO, 78.8 Ϯ 7.4. p ϭ 0.9. For 20°C versus 5°C: control, n ϭ 6, 94.0 Ϯ 4.5; CKO, n ϭ 6, 93.5 Ϯ 2.9. nϭ7,0.14Ϯ0.14.***pϽ0.05.B,ImmunostainingofPkconP21lumbarspinalcordsections p ϭ 0.4. at indicated genotypes. Insets, Pkc staining within the dorsal funiculus. C, Pkc␥ϩ neurons are derivedfromLbx1lineageneurons.DoublestainingofthenLacZprotein(green)andPkc␥(red) on P21 lumbar spinal cord sections from Tau-LSL-nlacZ;Lbx1-Cre fate mapping mice. D, Double requirement of Tlx3 activity beyond E12.5 in establishing this set staining of Pkc protein (red) with Tlx3 protein (green) on a P21 wild-type transverse lumbar of molecular identities. Transient Tlx1 expression is unable to spinal cord section. Arrow indicates colocalization; arrowhead indicates Pkc neurons lacking compensate this late Tlx3 activity, resulting in a loss of SOM and Tlx3. other markers at both lumbar and cervical levels (Fig. 2). Thus, early and late Tlx protein activities establish distinct molecular phenotypes as well as controlling transmitter receptor expression identities in dorsal horn excitatory neurons. (Qian et al., 2002; Cheng et al., 2004; Xu et al., 2008; Guo et al., 2012). Tlx1 is expressed in a subset of excitatory neurons of the Ontogeny of spinal neurons processing distinct cervical/thoracic spinal cord, and its expression is switched off sensory modalities once postmitotic neurons migrate out of the ventricular zone The dorsal horn excitatory neurons are divided into Lbx1 Ϫ class ϩ (Qian et al., 2002). In our CKO mice, Tlx3 expression was de- A neurons (dI1-dI3) and Lbx1 class B neurons (dI5 and dILB) tected normally at E12.5 but largely eliminated by E16.5. That is, (Figs. 1A and 8). Our studies and others show that this molecular expression of both Tlx1 and Tlx3 becomes transient throughout and developmental subdivision is correlated with distinct sensory the spinal cord in CKO mice. modalities processed by these spinal neurons. Two class A neu- A comparison of phenotypes of Tlx3 null versus CKO mice rons (dI1 and dI3) are located in the deep dorsal horn laminae provides insight into the temporal control of dorsal horn excit- and involved with sensory-motor coordination, with dI1 neu- atory neuron phenotypes. We found that transient Tlx3 expres- rons necessary for proprioception (Bermingham et al., 2001) and sion retained in CKO mice is sufficient to activate VGLUT2 in the dI3 neurons involved with hand grasp performance (Bui et al., dorsal horn, in contrast to complete loss of VGLUT2 expression 2013). The studies described here show that Tlx3-dependent class at the lumbar superficial dorsal horn in Tlx3 null mice (Cheng et B excitatory neurons (dI5 and/or dILB) are required to process al., 2004). The early determination of the glutamatergic transmit- pain-related and itch-related sensory information and to gener- ter phenotype is consistent with that VGLUT2 expression is ini- ate the escape response in response to dynamic mechanical stim- tiated shortly after birth of neurons and that transient Tlx1 uli (Fig. 8). expression in the cervical/thoracic dorsal horn is able to compen- Class B neurons can be further divided into two categories (I sate Tlx3 loss in establishing VGLUT2 expression (Cheng et al., and II), based on how their development is affected in Tlx3F/F; 2004). In contrast, expression of SOM, Tac1, GRP, GRPR, and Lbx1Cre/ϩ CKO mice. The first category (Fig. 8, “I”) includes PKC␥ in laminae I/II is eliminated in CKO mice, suggesting a those whose development is unaffected in CKO mice, such as the 14746 • J. Neurosci., September 11, 2013 • 33(37):14738–14748 Xu, Lopes et al. • Excitatory Spinal Neurons and Somatic Sensory Modalities

Figure 8. Ontogeny of spinal neurons processing distinct sensory modalities. I and II are referred to as two categories of Lbx1 ϩ class B excitatory neurons. Type I includes neurons whose development is unaffected in CKO mice, whereas Type II category is composed of a cohort of neurons whose development is impaired in CKO mice. Type II neurons are located in laminae I/II and are required to generate nocifensive motor behaviors evoked by pain-related or itch-related stimuli, as well as to generate escape response evoked by dynamic mechanical stimuli. Lbx1 Ϫ class A neurons mediate proprioceptive and mechanoreceptive sensory information. However, it is not clear whether the sense of innocuous cold or warm, which is preserved in CKO mice, is mediated by Lbx1 Ϫ class A neurons or by the Type I category of Lbx1 ϩ class B excitatory neurons (dashed arrows). SOM ϩ/S and Tac1 ϩ/S, SOM ϩ and Tac1 ϩ neurons at laminae I/II, or the superficial (“s”) laminae.

Phox2a ϩ subset of dI5 neurons and a portion of Tac1 ϩ neurons ing a concurrent requirement of DRG11/Prrxl1 for DRG neuron located in deep laminae. Genetic fate mapping shows that both development (Rebelo et al., 2006). Among type II class B neurons, Phox2a ϩ and Tac1 ϩ neurons do represent Lbx1 ϩ class B neu- GRPR ϩ and GRP ϩ neurons have already been implicated in pro- rons (Fig. 2). We reported previously that Tlx3 expression is cessing itch (Sun et al., 2009; Mishra and Hoon, 2013). The iden- switched off soon after the genesis of these neurons at E11.5 or tities of spinal neurons processing pain-related information are E12.5 (Qian et al., 2002; Xu et al., 2008). Accordingly, the pres- still poorly understood, but SOM ϩ and/or Tac1 ϩ neurons, rep- ervation of transient Tlx3 expression in CKO mice explains a resenting two large subsets of excitatory neurons located in lam- normal development of these neurons. Because the CKO mice inae I/II, could be attractive candidates. PKC␥ ϩ neurons respond retain the ability to sense innocuous cold and/or warm, we spec- to dynamic mechanical stimuli (Miraucourt et al., 2007; Neu- ulate that this type of somatic sensory information might be pro- mann et al., 2008); their developmental impairment might con- cessed by Type I category of class B excitatory neurons or by tribute to the loss of the touch-evoked escape response in CKO Lbx1-negative class A neurons, whose development is also unaf- mice. fected in CKO mice (Fig. 8, dashed arrows). Among spinal neurons responding to low threshold mechan- The second category (Fig. 8, “II”) of class B excitatory neurons ical stimuli, PKC␥ ϩ neurons are unique in terms of their location are those whose development is compromised in CKO mice, such in the inner layer of lamina II (Neumann et al., 2008), rather than as SOM ϩ, Tac1 ϩ, GRP ϩ, GRPR ϩ, and PKC␥ ϩ neurons located in the more conventional laminae III-V (Kandel et al., 2000; Lal- in laminae I and II. Genetic fate mapping shows that all these lemend and Ernfors, 2012). Here, we showed that these neurons neurons represent Lbx1 lineage neurons, with the exception of a are derived from Lbx1 ϩ class B neurons, rather than from Lbx1 Ϫ small subset of SOM ϩ neurons. Our behavioral analyses show class A neurons that mainly settle in laminae III-V (Helms and that this category of Tlx3-dependent neurons are specialized to Johnson, 2003). Moreover, upon central disinhibition after pe- process pain-related and itch-related information, as well as to ripheral nerve injury, PKC␥ ϩ neurons are part of the circuit that generate the touch-evoked escape response. An involvement of mediates pain evoked by low threshold mechanical stimuli Tlx3-dependent spinal neurons in sensing pain is consistent with (Takazawa and MacDermott, 2010). It should also be noted that that mice lacking DRG11/Prrxl1, a Tlx3-dependent gene (Qian et touch-evoked escape is a conserved behavior seen in worms, in- al., 2002), exhibited marked deficits in generating nocifensive sects, fishes, and mammals and likely evolves for animals to es- behavior in response to painful stimuli (Chen et al., 2001), al- cape from predators in the natural environment. Thus, the though the interpretation is complicated by a latter report show- shared developmental ontogeny of the type II class B excitatory Xu, Lopes et al. • Excitatory Spinal Neurons and Somatic Sensory Modalities J. Neurosci., September 11, 2013 • 33(37):14738–14748 • 14747 neurons appears to be correlated with the shared roles in sensing totic dI1 neurons of the developing spinal cord. Proc Natl Acad Sci U S A environmental danger and generating proper behavior to avoid 109:1566–1571. CrossRef Medline noxious stimuli or to escape from predators (Fig. 8). 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